A multiplex microsphere-based assay for the simultaneous detection of C. parvum, E. histolytica and G. lamblia antigen in human faecal sample

Abstract number: 1733_57

Groen J., Huwe A., Su X., Cox S., Truong H., Nguyen P., Laderman E.

Background:Cryptosporidium, Entamoeba, and Giardia are protozoan parasites that infect the gastrointestinal tract of animals and humans by oral-faecal transmission or through contaminated water sources. Infections may be asymptomatic or may cause a range of symptoms including diarrhoea, fever, and vomiting. Immunocompromised individuals are often unable to clear the parasites from their systems and ultimately suffer severe illness and possible death. Current methods of diagnosis include microscopy (O&P), and ELISA; these ``single-plex'' methods prove to be labour intensive, and require special skills in addition to other limitations. A high performance, multiplexed assay with the ability to simultaneously detect the presence of all three parasites in a single faecal sample is highly desirable. This study describes the development of the Plexus^TM Parasitic Multi-Analyte Diagnostics assay based on the Luminex xMAP^TM system (Austin, TX).

Methods: Samples: A retrospective panel of 248 human stool samples submitted for parasite testing was collected. In addition, stool samples, and culture supernatants of common enteric pathogens were collected for cross-reactivity testing. PLEXUS^TM Parasitic Multi-Analyte Multiplex Assay: Monoclonal antibodies specific for each parasite were covalently linked to microspheres. The capture microspheres were mixed with extracted human faecal samples, washed, and incubated with polyclonal detection antibodies specific for each parasite antigen. Finally, the microspheres were incubated with fluorescent Phycoerythrin (PE) conjugate. The median fluorescence intensity of PE measured by the Luminex 100 System indicates the amount of antigen captured. Reference Assay: Samples were tested by microscopy or with the TechLab^TMCryptosporidium II, E. histolytica II, and Giardia II ELISA systems per the manufacturer's suggested protocol.

Results: The sensitivity of the PLEXUS Parasitic Panel compared to ELISA was 100% (62/62) for C. parvum, 95.5% (21/22) for E. histolytica, and 98.4% for G. lamblia. The specificity was determined to be 100% (176/176) for all three parasites. In addition, the system does not display cross-reactivity with any of the common enteric pathogens, bacteria, or viruses.

Conclusion: The multiplex capability of the PLEXUS^TM Parasitic Multi-Analyte Diagnostics system offers a high performance, time-saving alternative to microscopy and ELISA for the qualitative detection of C. parvum, E. histolytica and G. lamblia.